Nuclear Histone 3 Post translational Modification Profiling in Whole Cells using Spectral Flow Cytometry

Data Analysis

Author

Serrano Lab

Background

In this analysis workflow we use spectral flow cytometry to simultaneously assess histone H3 post-translational modifications (H3-PTMs) at the single-cell level, in the context of cell cycle status and cell-type-specific markers. This workflow was implemented in BioProtocol Exchange by Golden et al., 2024 and contains supplementary figures and data analysis associated with the pre-print.

Information about biological material

iPSC-derived Neuronal Progenitor Cells (NPCs) were generated using the female control line SCTi003-A (Stem Cell Technologies). NPC induction was performed with the STEMdiff SMADi Neural Induction Kit, following the manufacturer’s instructions. All experiments were conducted with three biological replicates, defined in this context as NPCs induced in three independent wells. For further details on experimental design and data acquisition, please refer to Golden et al., 2024.

Information about initial data processing performed in OMIQ

Data processing Strategy Additional Information
Scaling Hyperbolic arcsine (arcsinh) Finak et al., 2010
Gating Cell QC and manual gating for comparison Golden et al., 2024
Subsetting Fully stained samples Removal of single color controls and blanks to prevent noise in downstream analysis
Subsampling Random downsampling. Equal number of cells available in the Live Cells filter Braanker et al., 2021 Note: Density-dependent downsampling strategy should be tested to assess if there is loss of small cell populations due to random cell selection Qiu, 2017
Dimension Reduction PCA all features
Dimension Reduction opt-SNE selected features
Clustering FlowSOM selected features

Data analysis and visualization

The goal of this analysis is to assess H3-PTM signatures in NPCs in the context of their cell cycle status and lineage markers (PAX6 in this case).

Show code
R version 4.4.1 (2024-06-14)
Platform: aarch64-apple-darwin20
Running under: macOS Ventura 13.2.1

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/New_York
tzcode source: internal

attached base packages:
[1] grid      stats     graphics  grDevices datasets  utils     methods  
[8] base     

other attached packages:
 [1] colorRamp2_0.1.0       statsExpressions_1.5.5 kableExtra_1.4.0      
 [4] patchwork_1.2.0        ggstatsplot_0.12.4     ggpubr_0.6.0          
 [7] stringr_1.5.1          xml2_1.3.6             downlit_0.4.4         
[10] svglite_2.1.3          Cairo_1.6-2            circlize_0.4.16       
[13] ggridges_0.5.6         wesanderson_0.3.7      dendextend_1.17.1     
[16] ComplexHeatmap_2.20.0  heatmaply_1.5.0        plotly_4.10.4         
[19] hrbrthemes_0.8.7       seriation_1.5.5        dendsort_0.3.4        
[22] readr_2.1.5            tidyr_1.3.1            ggExtra_0.10.1        
[25] lubridate_1.9.3        viridis_0.6.5          viridisLite_0.4.2     
[28] dplyr_1.1.4            ggplot2_3.5.1         

loaded via a namespace (and not attached):
  [1] RColorBrewer_1.1-3      rstudioapi_0.16.0       jsonlite_1.8.8         
  [4] shape_1.4.6.1           datawizard_0.12.2       correlation_0.8.5      
  [7] magrittr_2.0.3          SuppDists_1.1-9.7       farver_2.1.2           
 [10] rmarkdown_2.27          GlobalOptions_0.1.2     vctrs_0.6.5            
 [13] memoise_2.0.1           paletteer_1.6.0         effectsize_0.8.9       
 [16] rstatix_0.7.2           webshot_0.5.5           htmltools_0.5.8.1      
 [19] curl_5.2.1              broom_1.0.6             BWStest_0.2.3          
 [22] htmlwidgets_1.6.4       cachem_1.1.0            mime_0.12              
 [25] lifecycle_1.0.4         iterators_1.0.14        pkgconfig_2.0.3        
 [28] R6_2.5.1                fastmap_1.2.0           PMCMRplus_1.9.10       
 [31] shiny_1.9.0             clue_0.3-65             digest_0.6.36          
 [34] colorspace_2.1-1        rematch2_2.1.2          S4Vectors_0.42.1       
 [37] crosstalk_1.2.1         labeling_0.4.3          fansi_1.0.6            
 [40] timechange_0.3.0        httr_1.4.7              abind_1.4-5            
 [43] compiler_4.4.1          bit64_4.0.5             fontquiver_0.2.1       
 [46] withr_3.0.0             doParallel_1.0.17       backports_1.5.0        
 [49] carData_3.0-5           highr_0.11              Rttf2pt1_1.3.12        
 [52] ggsignif_0.6.4          MASS_7.3-61             rjson_0.2.21           
 [55] gfonts_0.2.0            tools_4.4.1             httpuv_1.6.15          
 [58] extrafontdb_1.0         glue_1.7.0              promises_1.3.0         
 [61] cluster_2.1.6           generics_0.1.3          gtable_0.3.5           
 [64] tzdb_0.4.0              ca_0.71.1               data.table_1.15.4      
 [67] hms_1.1.3               car_3.1-2               utf8_1.2.4             
 [70] BiocGenerics_0.50.0     ggrepel_0.9.5           foreach_1.5.2          
 [73] pillar_1.9.0            vroom_1.6.5             later_1.3.2            
 [76] gmp_0.7-4               bit_4.0.5               renv_1.0.7             
 [79] tidyselect_1.2.1        registry_0.5-1          fontLiberation_0.1.0   
 [82] miniUI_0.1.1.1          knitr_1.48              fontBitstreamVera_0.1.1
 [85] gridExtra_2.3           IRanges_2.38.1          crul_1.5.0             
 [88] stats4_4.4.1            xfun_0.46               matrixStats_1.3.0      
 [91] stringi_1.8.4           boot_1.3-30             lazyeval_0.2.2         
 [94] yaml_2.3.10             kSamples_1.2-10         evaluate_0.24.0        
 [97] codetools_0.2-20        httpcode_0.3.0          extrafont_0.19         
[100] gdtools_0.3.7           tibble_3.2.1            BiocManager_1.30.23    
[103] multcompView_0.1-10     cli_3.6.3               xtable_1.8-4           
[106] parameters_0.22.1       systemfonts_1.1.0       munsell_0.5.1          
[109] Rcpp_1.0.13             zeallot_0.1.0           png_0.1-8              
[112] parallel_4.4.1          assertthat_0.2.1        bayestestR_0.14.0      
[115] Rmpfr_0.9-5             mvtnorm_1.3-1           scales_1.3.0           
[118] insight_0.20.3          purrr_1.0.2             crayon_1.5.3           
[121] GetoptLong_1.0.5        rlang_1.1.4             TSP_1.2-4